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Pol Solans: Development of an Image J Macro for Analysis of Fluorescence Resonance Energy Transfer Experiments with Sensitized Emission Method.

Abstract

Fluorescence resonance energy transfer (FRET) is a process where an excited fluorescent molecule (donor) transfers its energy, in a non radiative way, to a close fluorescent molecule (acceptor). Sensitized emission method measures the increase of acceptor emission due to resonance energy transfer from the excited donor. In this method, crosstalk and crossexcitation factors are calculated to correct the FRET image (Fc). In order to compare experiments performed in different conditions or instruments, FRET efficiency can be calculated using a constant (G factor). G factor represents the ratio of the acceptor emission increase to donor fluorescence loss due to FRET. Here, we have developed a macro instruction for ImageJ, that allows to calculate in a friendly way: crosstalk and crossexcitation constants (a, b, c, d), G factor and FRET efficiency from sensitized emission FRET experiments (based on Vogel and Ikeda method). This macro allows also to calculate k factor, which is the ratio between the intensities of the donor and acceptor in the absence of FRET, when the stoichiometry of the pair is 1:1.

Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells. Chen H, Puhl HL 3rd, Koushik SV, Vogel SS, Ikeda SR. Biophys J. 2006 Sep 1;91(5):L39-41.

Keywords

FRET, Sensitized Emission Method

Administrative data

Presenting author: Pol Solans
Organisation: Universitat de Barcelona-IDIBAPS

co-authors: Anna Bosch, Neus Abella and Maria Calvo

Type: Poster (portrait)

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